A technique of rapidly growing use in laboratory manipulations of DNA is that of pulsed field electrophoresis. The technique was developed to resolve DNA fragments on the basis of size. In each of its many variations, this technique involves the switching of the electric field back and forth between two or more fields which differ in direction, to cause a periodic reorientation of the DNA strands. The degree of reorientation occurring within each pulse varies with the length of each strand, with the result that the net rate of movement of any particular strand in an overall direction of migration varies with the length of that strand, thereby permitting resolution according to strand length.
Early investigations of the technique include the frequently referenced patent of Cantor, et al., U.S. Pat. No. 4,473,452 (Sep. 25, 1984), and the companion paper, Schwartz, D. C., et al., "New Techniques For Purifying Large DNAs and Studying Their Properties and Packaging," Cold Spring Harbor Symposia on Quantitative Biology 47:189-195 (1983). The switching protocol disclosed by Cantor and Schwartz is between two electric fields in directions which are transverse (generally orthogonal) to each other, both in the plane of a slab gel, with intensity and duration differing between the two fields. The angle between the two fields changed as the DNA progressed along the gel, but the result is an overall migration direction which generally follows a line which bisects the angle between the two field directions. Variations of the technique include the use of angles other than 90.degree., e.g., 120.degree., and various refinements and adjustments for achieving control over field homogeneity. In a further variation of the technique, the two field directions are transverse to the plane of the gel as well as to each other, with the plane of the gel bisecting the angle between them. This variation is described by Gardiner, K., et al., Somatic Cell and Molecular Genetics 12(6): 185-195 (1986) and Gardiner, K., et al., Nature 331:371-2 (28 Jan. 1988), and is referred to as "transverse alternating field electrophoresis" (TAFE). Here, the two fields are of the same intensity and switching is done at equal time intervals, but again, the angle between the fields changes as the DNA migrates along the gel.
A second group of pulsed field techniques involve field inversion, or one-dimensional pulsing, rather than orthogonally oriented fields. In this group, known as "field inversion gel electrophoresis" (FIGE), the alternation is between two fields rotated 180.degree. with respect to each other rather than 90.degree., 120.degree. or any other transverse angle. A description of this technique is offered by Carle, G. F., et al., U.S. Pat. No. 4,737,251 (Apr. 12, 1988). According to the Carle, et al. method, net migration in one direction is achieved by an asymmetric field inversion profile, using either pulses of unequal duration, the forward pulse being longer than the reverse, or pulses of the same duration but unequal voltage gradient, the gradient in the forward direction being greater than that in the reverse.
A problem associated with FIGE techniques is band inversion, in which intermediate-size molecules migrate slower than both smaller and larger ones, the size vs. mobility curve turning back on itself, superimposing small molecules over large by giving both the same mobility. One attempt to overcome this problem is the technique known as "zero integrated field electrophoresis" (ZIFE), and is described by Turmel, C., et al., "High-Resolution Zero Integrated Field Electrophoresis of DNA," Electrophoresis of Large DNA Molecules: Theory and Applications, Cold Spring Harbor Laboratory Press, pp. 101-131 (1990). Here, the time-averaged voltage gradient E.sub.AV is defined by the following equation: EQU E.sub.AV =(E.sub.1 t.sub.1 -E.sub.2 t.sub.2)/(t.sub.1 +t.sub.2)
where E denotes the gradient (V/cm) and t the duration (sec), with the subscript 1 referring to the forward direction and 2 the reverse. Although the name given the technique by the authors denotes E.sub.AV equal to zero with E.sub.1 .noteq.E.sub.2 the preference of the authors is for E.sub.AV "slightly larger than zero," on the order of 0.33. This is said to eliminate band inversion for molecules of larger than 23 kbp in size.
These techniques have been developed for, and used almost entirely in, separations of double stranded DNA. For single stranded DNA, their use has met with limited success. Despite the ability of these techniques to improve the resolution of large double stranded DNA, they appear to offer little improvement for large single stranded DNA, where resolution is particularly difficult, if not lacking entirely, for fragments greater than 400 bases in length. The most widely used field inversion technique for single strand DNA has involved equal voltage gradients forward and reverse, with the forward duration exceeding the reverse. This results in a minor improvement in separating large single stranded DNA. It also results however in band inversion which, as in double stranded DNA separations, can obscure the separation. In addition, the lack of a flexible high voltage switching power supply has limited the investigation of the effects of pulsed field techniques on the separation of single stranded DNA. As a result, pulsed field electrophoresis has not been a viable technique for DNA sequencing or other separations involving single stranded DNA.
While fragments up to about 250 bases in length can be separated by conventional equipment and techniques, a variety of complex and unwieldy methods have been used for longer sequences. These include the use of gels which are longer than the conventional and commercially available sequencing gels (which are 40 cm to 80 cm in length), the use of voltage, buffer and salt gradients to vary the spacing of the bands along the length of the gel (neither of these methods gives a true improvement in resolution), the use of .sup.35 S isotope, the use of multiple lane sets loaded at different times to vary the duration of each run, the attachment of biotin, streptavidin or other large molecule (as reported by Ulanovsky, L., et al., "DNA trapping electrophoresis," Nature 343:190-192, 11 Jan. 1990), and methods by which the separated bands are moved continuously past a fixed detector such as an automated fluorescence detector.
An simple and effective method for separating single strand DNA which is effective over a wide range of strand lengths is needed. The present invention provides such a method and overcomes many of the problems of prior art techniques in a manner which demonstrates unexpected success.